MYCORRAY: a microarray tool for simultaneous detection of grapevine trunk fungi. Evaluation of the best DNA extraction method

Fungal pathogens causing grapevine decline and trunk diseases are widespread in different countries of the world causing significant reductions in vineyard longevity and productivity. Established vines can become infected through pruning wounds and during their lifetime can be re-infected multiple times with one or more causal agents of wood diseases. Therefore, it is common to isolate several pathogens from the discolored woody tissues of the trunk, including, among the most frequent, Phaeomoniella chlamydospora, several species of Phaeoacremonium and the Botryosphaeriaceae, Fomitiporia mediterranea and Eutypa lata. In order to set up a microarray tool (MYCORRAY) for simultaneous identification of the main fungal pathogens involved, a comparative evaluation of seven DNA extraction methods from grapevine sawdust collected by drilling the tissues surrounding pruning wounds of naturally infected vines, was carried out Measurements of total DNA concentration (A260) as well as real-time and end-point PCR amplifiability were used to determine quantity and quality of the extracted DNA. Meanwhile its suitability for indexing purposes was assessed by the mean of four independent nested-PCR assays specific for P. chlamydospora, Neofusicoccum parvum, E. lata and F. mediterranea, respectively. Probably due to the presence of PCR inhibitors that co-purified, total DNA concentrations were unsuitable for globally predicting PCR success. Meanwhile real-time PCR amplifiablity and relative Ct values were the best indicators of extract quality. However, when the presence of the targeted fungi was ascertained, not all methods were found to be equally suitable for detecting multiple infections of the wood.